This application from a collaboration between the Seufferheld and Blanke laboratories at the University of Illinois at Urbana-Champaign proposes studies to explore the overall hypothesis that Helicobacter pylori acidocalcisome-like granule (ALGs) are polyphosphate-rich organelles. Dense volutin granules have been previously described in the cytosol of H. pylori, which several lines of evidence suggest are acidocalcisomes, and which we tentatively describe as acidocalcisome-like granules (ALGs). Acidocalcisomes are membrane-bound organelles localized to the cytosol of several eukaryotic microorganisms. The recent discovery of acidocalcisomes in bacteria, in which the PI was the first to identify bacterial acidocalcisomes, is exciting because it is one of the first examples of a membrane bound organelle within bacteria, and supports an important functional role for acidocalcisomes across prokaryotic and eukaryotic kingdoms. The association of acidocalcisomes with the synthesis of polyphosphate (poly P) polymers has been established in eukaryotes. Poly P is associated with multiple functions in bacteria, and has important roles in bacterial pathogenesis for multiple pathogens, including the important human gastric pathogen H. pylori, the etiologic agent of several important gastric diseases. The capacity of H. pylori to successfully colonize and persist within the stomach requires that the organism survive the harsh conditions of this host niche. Notably, poly P has been demonstrated to be important for colonization of H. pylori within a murine infection model, as well as for stress resistance, in vitro. However, the mechanisms underlying this relationship between poly P and H. pylori stress resistance and virulence have not been studied, and remain poorly understood. Our long-term goal is to identify and characterize the mechanisms underlying poly P regulation and accumulation within H. pylori, as well as to understand the physiological role of ALGs and poly P in the physiology and pathogenesis of this important human pathogen. However, in order to reach these long-term goals in the future, it is first critical to validate that the dense volutin granules previously described in H. pylori are poly P-enriched ALGs. In this R03 application, we propose detailed studies to isolate and purify ALGs from H. pylori. We will evaluate the ALGs for known markers of acidocalcisomes and/or poly P metabolism. The studies supported by this R03 mechanism are important for establishing the experimental and conceptual framework for future studies to investigate the molecular composition and roles of ALGs in virulence for an important human pathogen. PUBLIC HEALTH RELEVANCE: Long-term infection with the human pathogen Helicobacter pylori is a significant risk factor for the development of gastric ulcer disease or gastric adenocarcinoma, one of the leading causes of cancer-related death worldwide. The completion of the research proposed within this application is critical for revealing fundamental new information about how this important human pathogen survives the harsh environment of the stomach, which in turn may reveal new targets for antibiotic development.